A troglobitic amphipod in the Ice Caves of the Shawangunk Ridge: Behavior and resistance to freezing

Subterranean Biology 15: 95—104 (2015)

doi: | 0.3897/subtbiol. 15.4733 RESEARCH ARTICLE LAD Dierranean

‘ O Published by
http://subtbiol.pensoft.net gy The Iertons Sey
for Subterranean Biology

A troglobitic amphipod in the Ice Caves of the
Shawangunk Ridge: Behavior and resistance to freezing

Luis Espinasa', Alex McCahill', Amber Kavanagh’,
Jordi Espinasa’, Alyssa M. Scott', Amy Cahill!

I School of Science, Marist College. Poughkeepsie, New York, USA 2. New Paltz High School. New Paltz, New
York, USA

Corresponding author: Luis Espinasa (luis.espinasa@marist.edu)

Academic editor: O. Moldovan | Received 15 February 2015 | Accepted 15 April 2015 | Published 10 June 2015
http://zoobank. org/8ADC7DED-AFEB-4C3 F-9991-BA360B33CE51

Citation: Espinasa L, McCahill A, Kavanagh A, Espinasa J, Scott AM, Cahill A (2015) A troglobitic amphipod in the Ice
Caves of the Shawangunk Ridge: Behavior and resistance to freezing. Subterranean Biology 15: 95-104. doi: 10.3897/
subtbiol.15.4733

Abstract

Stygobromus allegheniensis Holsinger, 1967 (Allegheny Cave Amphipod) is a troglobiotic crustacean com-
monly found in caves of the Northeast United States. We describe several new populations from the
unique tectonic Ice Caves found in the Shawangunk Ridge in New York, USA. Results also show that
despite being an eyeless species, it can detect particular wavelengths of light and individuals display scot-
ophilia, a preference for darkness. Finally, the Ice Caves pose a challenge to any aquatic troglobiont; in the
winter months, the Ice Caves freeze and the floor and walls become covered in solid ice. Our results show

that S. allegheniensis may seek warmer waters within the cave, but can also survive being frozen in solid ice.

Keywords
Stygobromus allegheniensis, amphipod, Shawangunk, scotophilia, cryoprotectant, Ice Caves, troglobite,
troglobiont

Introduction

Situated atop the the Shawangunk Ridge in the mid-Hudson Valley (New York, USA)
are the Ice Caves of Sam’s Point, Minnewaska, and Mohonk Preserves. These caves oc-
cur at the apexes of the broad anticlinal sections of the ridge where flat-lying, truncated

Copyright Luis Espinasa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

96 Luis Espinasa et al. / Subterranean Biology 15: 95-104 (2015)

beds of white Shawangunk conglomerate form cliffs. The Shawangunk Formation is a
silica-cemented conglomerate of white quartz pebbles and sandstone of extreme hard-
ness (Epstein and Epstein 1972). Unlike limestone, this type of rock does not easily
undergo the chemical weathering or mechanical erosion that leads to the formation
of solutional caves. On the contrary, the Ice Caves are an example of tectonic caves.
When the glacial ice sheets retreated at the end of the Pleistocene about 12,000 years
ago, the release of pressure from the melting of the thick overlaying ice generated a mass
movement of the bedrock. ‘The rocks separated along joints or fractures and were pulled
apart mechanically. The resulting caves are usually high, narrow fissures that have nearly
planar walls with matching patterns on opposite sides of the passage. The ceiling is often
a flat bed of rock that did not move or that moved along a different fracture. The mas-
sively bedded, widely jointed rock at the edge of the cliff breaks up into huge blocks that
tumble and lean against each other as they gradually work their way downslope.

Two circumstances potentially hinder these caves from being inhabited by
troglobionts. First, tectonic caves are typically small. The longest caves described
in this area are Ice Cave #1 (138 m) at Sam’s Point Preserve (Espinasa and Cahill
2011) and Xanadu Cave (422 m) at the edge of Mohonk Preserve (Millet and Boop
2013). Estimates of subterranean richness shows that larger caves accumulate species
at higher rates than smaller caves (Schneider and Culver 2004). The second hinder-
ing circumstance is that snow and cold air enter the caves through the openings at
the top and is then unable to escape. ‘This refrigerated environment often preserves
snow and ice into the summer. As they are aptly named, some of the Ice Caves’
walls and floors are covered with solid ice, blocking many of its chambers (Fig. 1).
Over the past few years in collaboration with the authorities at Sam’s Point Preserve,
The Nature Conservancy, and Mohonk Preserve, we have conducted studies of the
fauna that inhabit the Ice Caves. The most significant discovery is that despite the
aforementioned constraints, they are inhabited by an obligate cave-adapted aquatic
amphipod (Westlake 2009). Specimens of these crustaceans were identified by John
R. Holsinger, a taxonomic specialist of amphipods, as Stygobromus allegheniensis
Holsinger, 1967, the Allegheny Cave Amphipod. This taxonomic identification has
been corroborated by having identical histone sequences (GenBank# KP696361-
KP696363) to specimens from Clarksville Cave, NY (Cahill et al. 2015), where S. a/-
legheniensis has also been reported.

Stygobromus allegheniensis is fully depigmented, eyeless, and rather large, with some
individuals close to 2 cm long (Fig. 2). The species is found in caves of Maryland, Penn-
sylvania, and New York. The range of this troglobiont, one of the broadest of any in the
genus, covers a linear distance of approximately 596 km from north to south (Holsinger
1967). ‘The species is rather common in caves developed in the glaciated Appalachian
Plateau region. Despite its wide range, no significant morphological variation among
populations has been found, although this may be due to the general lack of large sam-
ples that have prevented a quantitative analysis of variation (Holsinger 1967).

In the Ice Caves, during the spring, summer, and fall water seeps through cracks
in the rocks and makes small streams and even pools of up tol10 m long and 2 m deep

A troglobitic amphipod in the Ice Caves of the Shawangunk Ridge... TP

Figure |. The Ice Caves at Sam’s Point Preserve, NY. Environmental conditions become challenging for

the survival of aquatic troglobionts during the winter months. A=B Entrance to Ice Cave #1 in summer (A)
and winter (B) C Deep lake in Ice Cave #2 during the Summer D Frozen floor in Ice Cave #3 on winter
E Walls in Ice Cave #1 become covered in ice during the winter, eventually blocking the passage to its

deeper chambers.

(Fig. 1C). These streams and pools are inhabited by scores of amphipods whose popu-
lations may exceed hundreds or even thousands of individuals (Espinasa and Cahill
2011). While specimens are mostly found in the dark areas of the Ice Caves, a few
have been found at the entrances where light illuminates the emerging streams. The
first objective of this study is to test the ability to detect light by this eyeless species.
The second objective concerns an interesting phenomenon. Each winter, much of the
Sam’s Point caves freeze over, possibly trapping the amphipods in the ice. How can
the amphipods survive this event? While the ability to survive in cold environments
has been well documented for many taxa, very few cave dwelling organisms have been
tested for their ability to seek warmer areas within the caves, or their ability to survive
exposure to subzero temperatures (Issartel et al. 2006; Novak et al. 2014). This is
probably because in typical limestone caves, temperature is for the most part constant
throughout the year and extreme fluctuations in temperature are not considered one
of the limiting factors influencing the life of troglobionts (Lencioni et al. 2010). Fur-
thermore, while in the cold limestone caves in the alpine and pre-alpine regions the

98 Luis Espinasa et al. / Subterranean Biology 15: 95-104 (2015)

Figure 2. Adult and juvenile specimens of Stygobromus allegheniensis from Ice Cave #1 at Sam’s Point
Preserve. Gravid females have been found in this cave, indicating that they are reproducing in this envi-
ronment. The species is fully depigmented, eyeless, with long appendages, typical of cave-adapted organ-
isms. During the warm months they can be found in the pools and small streams in the tectonic caves of

the Shawangunk Ridge.

internal temperature is generally lower than 10 °C, it rarely reaches zero or subzero
temperatures (Lencioni et al. 2010). Here we present data on an aquatic troglobite that
lives in tectonic caves that freeze over during winter.

Methods

Experiments were performed on 10/5/14 with 0.5—0.8 cm long specimens from Sam’s
Point Ice Cave #1. On that date, water temperature inside the cave was 7 °C. Three
sets of experiments were performed: Response to light, temperature preference, and
resistance to freezing. Specimens for each experiment were different and not reused.

Response to light

For the response to light, single individuals were put in a 62 cm x 1.5 cm x 1.5 cm
aluminum tank whose bottom was black and filled with water from the cave. Five

A troglobitic amphipod in the Ice Caves of the Shawangunk Ridge... 99

60 watt bulbs were placed 1 m away from the center of the tank. Half the tank was
covered with black plastic so as to have two 31 cm long sectors; one illuminated and
one dark. The specimen was left to acclimatize for 20 min in the tank. The time spent
by the individual in the illuminated side in the next 20 min (1200 sec) was recorded.
Four replicates with different individuals were performed. A two-tailed t-test was per-
formed to establish whether there was significant difference in time spent in either
side of the tank. At the end of the experiment, the temperature difference between the
two sectors was recorded and found not to be significantly different (Light=16.3 °C
+/- 0.35 Standard Deviation (SD); Dark=16.5 °C +/- 0.24 SD). The same protocol
was repeated, but instead of covering one sector with black plastic, it was covered with
green, yellow, and red translucent cellophane plastics to determine if the reaction to
light by these eyeless amphipods was modulated primarily by the detection of a specific
color of light.

Temperature preference

For determining temperature preference, 62 cm x 1.5 cm x 1.5 cm aluminum tanks
whose bottom was black and filled with water from the cave were used. A temperature
gradient was made within five of these tanks by having dry ice on one end of the tanks
and an electric heater pad on the other end. Amphipods could swim along the tem-
perature gradient within the tank. Lines were drawn on top of the tanks to subdivide
them into eight sectors of equal length. The first sector was completely frozen. The sec-
ond had an average temperature of 2.1 °C, and the following sectors had correspond-
ing temperatures of 7.6, 11.0, 14.4, 17.1, 20.2 and 21.5 °C (+/- 0.34 SD) as recorded
at the end of the experiment. One individual was deposited in each tank and left in
complete darkness for 20 min for acclimatization. While still in complete darkness
and with the help of a night vision Sony Digital 8 Handycam video camera, individual
tanks were observed every two minutes and the sections where the amphipods were
positioned at the end of the interval were recorded. A total of 100 data points were
recorded. A Kolmogorov-Smirnov test was performed to establish whether there was
significant difference in time spent in the sectors. At the end of the experiment when
the lights were turned on, it was noticed that the specimen from tank #5 crawled out
of the water, on top of the ice of sector one, and became frozen. When the ice melted
some minutes later, the specimen was alive and swam around normally.

Resistance to freezing

To examine the survival capabilities of the amphipods that experience freezing, experi-
ments were conducted in the field at Sam's Point Ice Cave #1 and in the laboratory. In
the field, individual specimens were deposited in a vial with about 5 ml of water. The
vial was then deposited in regular ice for 5 min and then transferred to dry ice until the

100 Luis Espinasa et al. / Subterranean Biology 15: 95-104 (2015)

water and specimen were frozen solid. Blocks of ice were melted after 5, 10, 15 and 60
min. The specimens in the laboratory were deposited in a 4 °C refrigerator for 24 hrs.
Individuals were then transferred to vials with about 5 ml of water and into a freezer
(-14 °C). As soon as there was a solid block of ice, the vials were immediately trans-
ferred to 0 °C so that the specimens did not have to endure temperatures significantly
lower than freezing. Blocks of ice with the amphipods were transferred to room tem-
perature to thaw after 1, 2, 3 and 12 hrs and the specimens were observed for survival.

Results and discussion

The presence of S. allegheniensis within the Shawangunk Ridge represents a new locality
and an extension of the range for the species. Previous reports are mostly for limestone
caves. Specimens of S. allegheniensis have been found during this study in the Ice Caves
located on the eastern cliffs of Sam’s Point, Ulster Co (N 41°40'19", W 74°20'47" 610
masl) and at Xanadu Cave in Table Rock at the edge of Mohonk Preserve, Ulster Co
(N 41°48'55", W 74°06'45" 120 masl). These localities are of particular interest as they
show the potential of the species to form large populations within the comparatively
small tectonic fissures and caves of the Shawangunk Conglomerate.

Our results also indicate that S. allegheniensis, despite being eyeless, detects light,
and exhibits scotophilia (preference for darkness). Specimens in our experimental
conditions preferred the dark side of the tanks, spending only an average of 9.9% of
the time (119 seconds +/- 142 SD out of the allotted 1,200 seconds; .01>P>.005)
in the illuminated side. When the black top covering half of the tank is replaced by
a translucent colored cellophane filter, it was noticed that the specimens drastically
change their behavior depending on the color of the light received. When the filter
was yellow or red, specimens reacted in a similar way as if that half of the tank was
in the dark. Out of 1,200 seconds, they spent 3.5% (42 seconds +/- 68 SD; .001>P)
and 20% (246 seconds +/- 215 SD; .05>P>.02) in the fully illuminated side. On the
contrary, when half the tank was covered by a green filter, specimens did not appear
to prefer the covered side, spending 72% (874 seconds +/- 545; .50>P>.20) in the
fully illuminated side.

When the amphipods were presented with a temperature gradient spanning from
0—21.5 °C, all five specimens had a clear preference (.001>P) for a temperature close to
14.4 °C (Fig. 3). Of the seven sectors available for swimming, specimens spent 40% of
the time in the sector with water at 14.4 °C followed by 25% of the time in the sector
at 11.0 °C. Both temperatures are higher than the temperature of the water in the cave
(7 °C) at the time specimens were collected. It is worth noting that while they clearly
had an immediate aversion to temperatures higher than about 18.5 °C, they did not
react so strongly to low temperatures (Fig. 3). Specimens were seen walking for short
periods directly on the ice and one even allowed itself to be encased in the ice rather
than swim to warmer water. While the event occurred at the end of the experiment,
when lights were on and the observer might have been generating some noise, it is still

A troglobitic amphipod in the Ice Caves of the Shawangunk Ridge... 101

Temperature preference
50

all |

2.1 7.6 11.0 14.4 17.1 20.2 21.5
Temp °C

% of time
i) W A
> ) © So

="
©

Figure 3. Temperature preference of the amphipods inhabiting Ice Cave #1. When presented with a
water temperature gradient, specimens spent most of the time swimming in water at 14.4 °C. Specimens
showed a strong and immediate aversion to temperatures higher than about 18.5 °C, but not to low tem-

peratures, with specimens walking directly on ice.

interesting that specimens would allow themselves to become encased in ice, especially
considering these amphipods move quite fast even in water close to 0 °C and could have
easily avoided it. When the ice was thawed, the specimen was alive and swam normally.

Specimens were frozen in the field inside a block of ice for up to 15 min and were
still alive and moving normally when the ice was thawed. In the laboratory, where
specimens were subjected to 24 hrs at 4 °C before being frozen solid, specimens sur-
vived being frozen in a block of ice for up to 2 hrs (Fig. 4). A movie clip of the proce-
dure can be seen at https://www.youtube.com/watch?v=Mgaj TnWVI13s.

Conclusions

We report here a new series of localities where the amphipod Stygobromus allegheniensis
can be found; the Ice Caves of the Shawangunk Ridge. Most previous reports for this
species are from limestone caves. The new localities are from tectonic caves, which are
comparatively smaller as they tend to be simple faults and cracks in the bedrock. These
Ice Caves are also unique in being covered with ice even during spring. During the
warmer months, some of the Ice Caves have small streams and even some 2 m deep
pools where the amphipods can be found in great numbers.

Our results show that S. allegheniensis, despite being eyeless, can still detect light
and shows scotophilia. Since the species can be found at the entrance of the caves,
but needs to seek dark environments for its survival, it is easy to understand why this

102 Luis Espinasa et al. / Subterranean Biology 15: 95-104 (2015)

RENT
rate le ene
a A

4

Figure 4. Specimen of S. allegheniensis frozen in a solid block of ice. Specimens were alive and behaved

normally when thawed up to two hours after being frozen.

ability has been maintained. Future research should address why light detection is re-
stricted to certain colors of light and what cells/organs are used for detection in these
otherwise eyeless species.

The winter conditions of the Ice Caves pose an interesting question as to how
these aquatic amphipods can survive the subzero temperatures and the coating of ice
on the walls and floors of these caves. Based on our data, we propose two possible
explanations. The first is that the deep pools present in the caves may not freeze
entirely, just their surface, leaving a safe haven where the population can survive. As
a deep pool freezes over, the amphipods would most likely remain at the bottom to
avoid being frozen. Our results regarding preference for water at around 14 °C sug-
gest that amphipods may actively move and seek the warmer, non-freezing areas. In

A troglobitic amphipod in the Ice Caves of the Shawangunk Ridge... 103

the winter, ice blocking the passages that lead to these pools has prevented us from
confirming this hypothesis, but should be a priority of future studies. The second ex-
planation is that the amphipods may have the ability to survive being frozen for long
periods of time in a hibernation-like state. Our results showed that amphipods can
survive being frozen. Although the maximum amount of time amphipods survived
in a solid block of ice in the laboratory was 2 hours, the procedure does not faithfully
replicate the actual process of how the amphipods become frozen in the Ice Caves.
In the field, it is most likely a progressive cooling and freezing since the temperature
would show a slow decrease over weeks, allowing their metabolism to adjust accord-
ingly. It is thus likely that in the field they are better able to survive freezing for much
longer than 2 hours.

Such a process has been documented in the aquatic subterranean amphipod
Niphargus rhenorhodanensis (Issartel et al. 2006). Cold acclimation induced an increase
in the crystallization temperature values but no survival was observed after thawing.
However, after inoculation at high sub-zero temperatures, cold-acclimated NV. rhe-
norhodanensis survived. The accumulation of cryoprotective molecules such as glycerol
(Issartel et al. 2006) and free amino acids (Colson-Proch 2009) may be linked to the
survival of this species when this species was cold-acclimated. It may be that S. al
legheniensis is also capable of undergoing equivalent metabolic adaptations in the cold
environment encountered in the Ice Caves.

Our results already show the species can be kept alive in the laboratory for ex-
tended periods and has many interesting characteristics worthy of study. As such, it is
likely that in the future the species will become a valuable model on which much more
research concerning troglobites can be performed.

Acknowledgments

Partial support for the project came from the Cleveland Grotto Science Fund grant
to AM and AK, from a National Speleological Society education grant to LE, and
from the School of Science at Marist College. Heidi Wagner, Sam’s Point Preserve
Manager, was instrumental in the development of the project. Her enthusiasm,
kindness, and support are greatly thanked. She even went caving to show us a cave
with one of the deep pools. Collecting permits were obtained with the help of the
following persons: Cara Gentry and Heidi Wagner (The Nature Conservancy, Sam’s
Point Preserve); John E. Thompson (Daniel Smiley Research Center, Mohonk Pre-
serve); Laurence Davis, Thom Engel, Michael Chu, and Chuck Porter (Northeast-
ern Cave Conservancy, Clarksville Cave Preserve). Identification of the species was
performed by John R. Holsinger. Field and laboratory studies were done with the
help of students from the Fall 2014 course of BIOL192: Field Biology at Marist
College and Emily Collins. Finally, a thank you to Monika Espinasa for reviewing
the manuscript.

104 Luis Espinasa et al. / Subterranean Biology 15: 95-104 (2015)

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